software for degenerate PCR primers

Jim Owens jow at helix.nih.gov
Tue May 25 08:31:08 EST 1993


In article <1993May21.171649.5657 at midway.uchicago.edu> steven joseph
chmura, sjchmura at kimbark.uchicago.edu writes:
>I am attempting to use RT-PCR to dectect low levels of mRNA expression.
>So far I have created a huge mess.  I have non-specific binding problems
>and high bckground noise.  I am using the GIBCO BRL SuperScript kit.

I have been using BRL's M-MLV reverse transcriptase.  You want some
RNaseH activity to cut up the RNA after it is reverse transcribed (see
BRL-Focus 13:26-29 [1991]).

Also, be sure to destroy the RTase (5 min, 95 degrees C) before the PCR
since RTase seems to bugger up Taq Polymerase. (Sellner et al, Nucl Acids
Res 20: 1487-1490 [1992])

Watch your buffers as you go from RTase to PCR.  The MgCl2 in the 5x
RTase buffer supplied by BRL will make the RTase reaction 5mM; diluted
1:4 as I do for PCR means 1.25mM MgCl2 without addition of any more Mg++
in the PCR buffer.  I have found that RTase will work with BRL's buffer
diluted 1:7.5 as well as 1:5.  Then a 25microliter RTase reaction will
only add 0.5mM Mg++ to a 100microliter PCR.

>
>Conditions:
>
>Primers for RT and PCR: 20bp 130 bp apart
>PCR conditions :
>	94 at 1min
>	50 at 2,72 at 2 min
>	72 at 7 min
>
>The mRNA was extracted from liver tissue.

Is the RNA in good shape?

>
>I am wondering the following:
>
>Would RNase free DNase be a wise treatment before the RT reaction
>to minimize genomic DNA contamination?

I don't know since I've never needed to worry about it.  I use two
negative controls: one, without RTase (to detect DNA), and one, without
RNA (to detect carryover of products from previous PCRs).

>
>Make the PRimers longer?

I favor longer primers, 23 to 25 bases, but they require higher annealing
temperatures.

>
>Change the PCR annealing times?


Times look more than enough, given the length you are expecting.  I have
been able to generate cDNAs that are over 1 kb long.  Unfortunately I
have not been able to generate 1 kb and longer PCR fragments (like I can
using genomic DNA), but I do get shorter PCR fragments from all along the
cDNAs.

Good luck,

Jim Owens



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